The site of a glycoprotein where an N-linked glycan chain binds can be predicted from a genetically encoded amino acid sequence, and those amino acids are referred to as an N-linked glycosylation site. In the case a protein having an ER localization signal in the manner of secretory proteins or membrane proteins has this N-linked glycosylation site, it has the possibility of being subjected to N-linked glycosylation. However, this does not mean that N-linked glycan chains are linked to all the N-linked glycosylation sites of a protein, and even in the case of the same N-linked glycosylation site of the same protein, the state (presence or absence) of the N-linked glycan chain addition may differ according to the molecule, and the state of an N-linked glycan chain addition is thought to be dependent on the conformation of the protein or type of expressing cells and the like.
Until now, several methods have been reported for investigating a site where an N-linked glycan chain is actually linked to an N-linked glycosylation site (to also be referred to an “N-linked glycosylated site”), and the N-linked glycosylated sites of various glycoproteins have been determined.
In Non-Patent Document 1, N-linked glycosylation and the structures of N-linked glycan chains at N-linked glycosylation sites were predicted during the course of determining the secondary structure of gp120, a structural protein of HIV. That method consisted of subjecting reductively alkylated gp120 glycoprotein to deglycosylation treatment with peptide N-glycanase F having diamidase activity (to be referred to as “PNGase F”) or EndoH, which is a type of endo-type glycosidase, further digesting with trypsin or Asp-N and the like, isolating by reverse-phase chromatography, comparing the resulting chromatogram with a chromatogram obtained by digesting a non-deglycosylated glycoprotein with trypsin or Asp-N and the like, and judging a peptide having a prolonged elution time to have been removed of the linked N-linked glycan chain, namely judging that an N-linked glycan chain had been linked to that peptide. Non-Patent Document 1 does not disclose or suggest digesting a protein that has the same amino acid sequence as gp120 protein but does not have a glycan chain with Asp-N and the like followed by comparing the resulting peptide fragment, nor does it disclose or suggest determining the addition of an N-linked glycan chain to an Asn residue by utilizing the change of an asparagine residue having a glycan chain linked thereto to an aspartic acid residue when subjected to deglycosylation treatment with an enzyme having deamidase activity.
In addition, in Non-Patent Document 2, after carrying out fragmentation on a glycoprotein, the glycosylated peptide is recovered with lectin followed by subjecting to deglycosylation treatment with PNGase F and analyzing by LC/MS. However, there is no disclosure or suggestion whatsoever regarding specifically hydrolyzing the glycoprotein according to an Asn residue or aspartic acid residue (Asp residue).
In addition, in Non-Patent Document 3, a method for determining a site where an N-linked glycan chain has actually been linked is indicated that consists of removing an amino group of an Asn residue when subjecting a glycoprotein to deglycosylation treatment with PNGase F having deamidase activity, and utilizing the change to an Asp residue as a result thereof. More specifically, in this method, in the case a peptide fragment, obtained by deglycosylation treatment of N-linked glycan chain of a glycoprotein by PNGase F followed by fragmenting with a peptidase such as trypsin, contains an N-linked glycosylation site and has an N-linked glycan chain linked to that site, the site where an N-linked glycan chain has been linked is determined by using as an indicator an increase of one dalton in the mass of the fragment from the theoretical value thereof. However, the method of Non-Patent Document 3 does not use an enzyme that specifically hydrolyzes an Asn residue or Asp residue. In addition, this method is an effective method for determining the site were an N-linked glycan chain is actually linked in the case of a uniform addition state of N-linked glycan chains at an N-linked glycosylation site. However, in the case N-linked glycosylation is not uniform, two fragments differing in mass by one dalton may be detected in the case the resulting peptide fragment has the original Asn residue and the case in which it has changed to an Asp residue. During actual measurements, it is necessary to consider the effect of naturally-occurring isotopes, and in the case two masses are detected that differ by one dalton, it is extremely difficult to clearly distinguish between whether the difference in mass is due to the naturally-occurring isotope or a difference of one dalton attributable to the difference in mass between the Asn residue and Asp residue.